calpain and zinc-chloride

BAK/BAX-mediated mitochondrial outer-membrane permeabilization (MOMP) drives cell death during development and tissue homeostasis from zebrafish to humans. In most cancers, this pathway is inhibited by BCL-2 family antiapoptotic members, which bind and block the action of proapoptotic BCL proteins. We report the 1.5 A crystal structure of calpain-proteolysed BAK, cBAK, to reveal a zinc binding site that regulates its activity via homodimerization. cBAK contains an occluded BH3 peptide binding pocket that binds a BID BH3 peptide only weakly . Nonetheless, cBAK requires activation by truncated BID to induce cytochrome c release in mitochondria isolated from bak/bax double-knockout mouse embryonic fibroblasts. The BAK-mediated MOMP is inhibited by low micromolar zinc levels. This inhibition is alleviated by mutation of the zinc-coordination site in BAK. Our results link directly the antiapoptotic effects of zinc to BAK.

Topics: Amino Acid Sequence; Animals; Apoptosis; bcl-2 Homologous Antagonist-Killer Protein; Binding Sites; Calpain; Chlorides; Crystallography, X-Ray; Humans; KB Cells; Magnetic Resonance Spectroscopy; Mice; Models, Molecular; Molecular Sequence Data; Peptide Fragments; Protein Conformation; Protein Structure, Secondary; Proto-Oncogene Proteins; Recombinant Proteins; Sensitivity and Specificity; Zinc Compounds

A study was conducted to examine the effects that physiological levels of m-calpain (calpain requiring millimolar concentrations of Ca2+) extract and a lysosomal extract have on myofibrillar proteins in vitro, and the effects that zinc has on inhibiting proteolysis by these extracts. During a 22-h incubation period, the lysosomal extract degraded myosin heavy chain, alpha-actinin, desmin, troponin-I, and myosin light chains 1 and 2. The effectiveness of the lysosomal extract to degrade myofibrillar proteins was significantly affected by the presence or absence of EDTA. Zinc, which is a potent inhibitor of cysteine proteinases, prevented most, but not all, of the lysosomal extract-induced myofibrillar protein degradation. Incubation of myofibrils with m-calpain resulted in the hydrolysis of troponin-T, desmin, and a 58-kDa molecular weight protein, possibly vimentin, and 5 mM ZnCl2 completely blocked these changes. Results from this study indicate that the degradation by the lysosomal extract is far more extensive than the degradation that occurs with normal postmortem storage and that possibly a non-cysteine protease is present that is capable of hydrolyzing some myofibrillar proteins under this in vitro condition, because Zn2+ did not block all proteolysis. However, similar changes were induced by m-calpain incubation and postmortem storage.

Topics: Animals; Calpain; Chlorides; Culture Techniques; Hydrolysis; Lysosomes; Muscle Proteins; Myofibrils; Postmortem Changes; Sheep; Time Factors; Zinc; Zinc Compounds